Angiotensin II (AngII) is a major hormone responsible for maintenance of electrolyte balance and blood pressure when the extracellular volume drops due to sodium depletion. One of its sites of action is the proximal tubule of the kidney where AngII at pM concentrations stimulates Na bicarbonate and fluid reabsorption while in the muM range it decreases NaCI reabsorption. New, important aspects of AngII regulation in the proximal tubule have recently become apparent due to the discoveries of AngII receptor subtypes (AT1A; AT1B; AT; non-AT1-non-AT2), of a novel signaling pathway involving arachidonic acid, and of the presence of CFTR and CI conductance. This project is directed towards understanding the signaling pathways and specific electrolyte transporters associated with different AngII receptor subtypes. The specific aims include:10 An evaluation of the effect of agonists/antagonists of AT1A AT1B, and AngIII/AngIV (non-AT1-non- AT2) receptors on cytosolic Ca levels and proton fluxes across the brush border and basolateral plasma membrane. 2) Determination of the importance of the cytochrome P450 system for arachidonic acid-dependent signaling in the proximal tubule. 3) Evaluation of the importance of luminal CI conductance and the Cystic Fibrosis Transmembrane Regulatory (CFTR) protein. Cytosolic Ca levels and pH will be measured with ion-sensitive fluorescent dyes and video microscopy. The role of P450 2CAA will be assessed by correlation of transport responses to AngII with immunological/biochemical analysis of enzyme levels and arachidonic acid metabolism before and after transfection of the very similar enzyme P450 2C2. CI conductance will be assessed by electrophysiological techniques and correlated with levels of CFTR message and protein. One of the important tools available for these studies are proximal tubule cell lines that we recently established. These cell lines are suitable for both biochemical, molecular biological, and cell-physiological investigations and thus should help to bridge the gap between information about receptors/signaling pathways and electrolyte transport. These studies should provide significant information and insights into the importance of different luminal and basolateral AngII receptors for proximal tubular electrolyte transporT.